T-cell-mediated cytotoxicity has been generated by culturing immune spleen cells from untreated donors without the addition of any known cellular antigens. The effectors generated preferentially lysed H-2 matched targets, but also lysed to some extent allogeneic target cells. High (H-2d) and low (H-2K,b) responder strains were identified, and strong responsiveness was a dominant characteristic. Specificity studies indicated that multiple clones of cytotoxic cells were generated in these cultures. Separate clones of effector cells could be identified which were responsible for the H-2 restricted and the non-restricted components. The findings suggest that the self-restricted component may be directed in part at either components present in a specific serum (i.e. fetal calf but not horse) or antigens induced by that serum.